This invention relates to the determination of the glucosylated form of hemoglobin known as Hb A.sub.1c in human blood samples. The determination of the extent of glucosylation of hemoglobin in an individual's blood provides a useful index of glucose level control in diabetics. In particular, the present invention concerns the preparation of monoclonal antibodies which recognize specifically the glucosylated N-terminal peptide residue in such human hemoglobin.
Patients afflicted with diabetes are incapable of metabolizing glucose in a conventional manner resulting in a build-up of glucose in their blood and urine. Conventionally, the glucose level in such body fluids is taken as a measure of the state of the diabetic condition which, in turn, is used as a guide for the amount of insulin or other agent to be taken or of the need to change the patient's diet.
This works moderately well except that the glucose level may fluctuate widely in dependence upon the time and content of the last meal, the last insulin injection, and the like. Thus, the reading will reflect an instantaneous condition which might not truly identify the longer term state of the diabetic condition. To circumvent the single glucose determination, more elaborate measurements (e.g., the 4 to 8 hour glucose tolerance tests) are used to measure the blood levels of glucose following an oral administration. These latter tests are time consuming, expensive and the individual must fast during the course of the assay.
It is known that another effect of the diabetic condition is an increase in the amount of glucosylated hemoglobin (Hb) in the blood of the diabetic. Hemoglobin is a protein tetramer made up of four chains (subunits) of amino acids, each of about 143 units and having a total molecular weight of about 64,000. At one end of the molecule (the NH.sub.2 -terminus of the beta-subunit) there is a valine unit which can react with glucose. The glucosylation of hemoglobin occurs by a non-enzymatic reaction involving glucose and the alpha-amino group of valine. Following a Schiff base formation between the reactants, the glucose undergoes an Amadori rearrangement forming 1-deoxyfructovaline. This complex is covalent and is not reversible. The glucosylation reaction is governed by the concentration of the reactants, e.g., hemoglobin and glucose. In a normal (non-diabetic) individual approximately 3% of the total hemoglobin is glucosylated. Hemoglobin tetramers with a 1-deoxyfructo-valine on the N-terminus of a beta-chain are identified as being glucosylated or A.sub.1c hemoglobin.
Glucose levels in diabetics are sufficiently high to increase the rate of glucosylation in direct dependence upon the glucose level in the blood, which reflects the severity of the diabetic condition. With hemoglobin, the A.sub.1c level is raised to about 5 to 12%. Since the circulating life span of hemoglobin is about 120 days, a glucosylated hemoglobin measurement will give a value which reflects an average glucose level for that period. Notably a meal high in glucose will not be reflected in a high glucosylated hemoglobin or serum albumin level. Thus, measurement of the glucosylated hemoglobin content gives a truer picture of the average circulating glucose levels and thus a truer picture of the long term condition of the patient.
One way of using this measurement has involved passing a lysed blood sample through a boronate column, thereby selectively adsorbing the A.sub.1c fraction of the hemoglobin along with some other undesired glucosylated non-specific components. The column is washed and the A.sub.1c determined spectrophotometrically. The process is complex, time consuming, and temperature dependent, and sometimes gives variable results.
An alternative analytical method involved subjecting a lysed blood sample to electrophoresis but electrophoresis is slow and expensive and requires considerable operator skill so the test is not practical for a clinical laboratory.
Dixon in Biochem. J.(1972)129,203-208 reacted glucose with valylhistidine, but solely for experimental purposes, no utility being expressed for the product.
U.S. Pat. 4,247,533 discloses an analytical technique wherein antibodies to Hb A.sub.1c were reportedly raised in a special sheep by injection of Hb A.sub.1c and absorbed with nonglucosylated hemoglobin to provide polyclonal antibodies which distinguished between Hb A.sub.1c and nonglucosylated Hb. Such antibodies then form the basis for a test to determine the proportion of glucosylated hemoglobin in a sample. The test, however, requires an appropriately immunized sheep and antibody absorptions to attain the proper specificity. It is, therefore, costly and difficult to produce specific polyclonal antibodies. The antibody preparations produced by this absorption approach are reported to be of low titer and affinity. The reproducibility of this approach is also open to question since there are no recent reports describing its use for the analysis of clinical samples of human hemoglobin.
Another attempt to obtain antibodies specific for Hb A.sub.1c is found in U.S. Pat. No. 4,478,744. The workers in U.S. Pat. No. 4,478,744 substituted a synthetic peptide immunogen for the normal hemoglobin molecule as the immunizing agent injected into an animal which normally does not have Hb A.sub.1c in its bloodstream, e.g., sheep. The synthetic peptide immunogen comprised a glucosylated peptide residue having an amino acid sequence corresponding to between the first 4 to 10 amino acids in the N-terminal hemoglobin sequence. Subsequent investigations, reported hereinbelow, have found that the sheep polyclonal antiserum raised against the synthetic peptide immunogen has no detectable specificity for the glucosylated form, Hb A.sub.1c.
______________________________________ Definitions Amino Acid Abbreviation ______________________________________ Arginine Arg Aspartic Acid Asp Glutamic Acid Glu Lysine Lys Serine Ser Asparagine Asn Glutamine Gln Glycine Gly Proline Pro Threonine Thr Alanine Ala Histidine His Cysteine Cyr Methionine Met Valine Val Isolencine Ile Leucine Leu Tyrosine Tyr Phenylalanine Phe Tryptophan Trp Alpha-Aminobutyric Acid Aba ______________________________________